Gaucher's disease is an autosomal recessive lysosomal storage disease which results from a deficiency in the lysosomal enzyme, glucocerebrosidase. This causes the accumulation of glucoceramide within peripheral macrophages, resulting in cellular enlargements. Gaucher's disease causes spleen, liver and bone marrow to malfunction and deteriorate.
Ceredase is the commercial name for glucocerebrosidase derived from human placenta. The intravenous infusion of Ceredase was shown to be clinically useful and has been approved for the treatment of Gaucher's disease in a study published in 1991 (Proc. Natl. Acad. Sci. 1990, 1913-1916). In order for the enzyme to be taken up by the target cell type, the enzyme is first deglycosylated, resulting in a mannose-terminated glycan structure so that it is taken up into the macrophage via its lectin receptors. This placental derived glucocerebrosidase treatment was approved by the Federal Drug Administration in April, 1991. However, due to the anticipated demand for this new replacement enzyme therapy, ways to produce this protein through means of recombinant expression have been sought.
Cerezyme is a recombinant form of glucocerebrosidase produced in CHO cells. The expressed protein is treated with exoglucosidases to produce the mannose sugars on the terminus of the existing polysaccharide chain, leading to a selective uptake of the enzyme by macrophages that are present in liver, spleen and skeleton. The drug works as well as the naturally occurring protein does by catalyzing the hydrolysis of the glycolipid glucocerebroside to glucose and ceramide as part of the normal degradation pathway for membrane lipids. However, the method of production of Cerezyme involves extensive treatment with exoglucosidases to trim the glycan structures to terminal mannose structures. This process is difficult to control and adds to the cost of production.
Major antigen presenting cells, such as dendritic cells and macrophages, express mannose-biding C-type lectins on their surface. Recombinant glycoproteins with mannose-terminated N-glycans are capable of specifically binding to and targeting such dendritic cells and macrophages via the mannose-binding C-type lectin on macrophages. Accordingly, it is desired for recombinant proteins which are intended for use as vaccines to comprise mannose-terminated N-glycans. The same problems as described above for production of Cerezyme exist in relation to methods of production of other recombinant glycoproteins with mannose-terminated N-glycans for use, e.g., in vaccines.
There therefore exists a need in the art for a method of production of glucocerebrosidase and other recombinant glycoproteins with mannose-terminated N-glycans which avoids such post-expression processing.
One possibility is to make use of CHO cells which have mutations in the post-translational glycosylation pathway. Van Patten et al. (2007, Glycobiology, 17, 467-478) describes a study carried out to examine the use of alternative expression systems to produce Cerezyme (recombinant glucocerebrosidase) that would circumvent the need to use exoglucosidases to trim the glycan structures to terminal mannose structures. The alternative expression systems tested included non-mammalian expression systems, CHO cells cultured in kifunensine and a CHO glycosylation mutant, Lec 1.
The study showed that these different expression systems could indeed produce glucocerebrosidase containing terminal mannose residues. Glucocerebrosidase produced by Lec 1 cells was in particular found to function just as well as the commercially available Cerezyme. However, the study also identified a number of significant issues with each alternative.
With regard to use of Lec 1 cells, Van Patten et al. (2007, Glycobiology, 17, 467-478) found that both this expression system was characterised by low productivity. Given this, it would be difficult, if not impossible, to scale up expression to industrial production levels.
Furthermore, Lec 1 cells are not amenable to large scale culture. Lec 1 cells were originally isolated from a proline auxotroph for genetic studies and do not survive very well in normal large scale cell culture conditions or in chemically defined media and cultured in suspension. Furthermore, the media has to supplemented with proline and even then the cells die easily. Thus this cell line is not robust and therefore not suitable for use in the production of recombinant proteins as their expected growth characteristics and productivity is inferior to existing industrial cell lines.
There therefore exists a need in the art for a scalable method of production of glucocerebrosidase and other recombinant glycoproteins with mannose-terminated N-glycans that avoid that avoids these problems of the prior art.